A human ribosomal protein S3 (rpS3), which also functions as a DNA repair enzyme(UV endonuclease III), was expressed in a methylotrophic yeast, Pichia pastoris, and biochemically characterized. UV endonuclease activity was preiously characterized, and this activity of mammalian rpS3 was found...
A human ribosomal protein S3 (rpS3), which also functions as a DNA repair enzyme(UV endonuclease III), was expressed in a methylotrophic yeast, Pichia pastoris, and biochemically characterized. UV endonuclease activity was preiously characterized, and this activity of mammalian rpS3 was found to be non-specfic upon purification and storage. Under the Pichia expression system, the subcloned cDNA of the human rpS3 gene revealed a peptide of 42 kDa by SDS-PAGE and Western blot. The secreted form of human rpS3 rendered no endonuclease activity while the intracellular form showed UV specific endonuclease activity by the nick circle assay.
A human ribosomal protein S3 (rpS3), which also functions as a DNA repair enzyme(UV endonuclease III), was expressed in a methylotrophic yeast, Pichia pastoris, and biochemically characterized. UV endonuclease activity was preiously characterized, and this activity of mammalian rpS3 was found to be non-specfic upon purification and storage. Under the Pichia expression system, the subcloned cDNA of the human rpS3 gene revealed a peptide of 42 kDa by SDS-PAGE and Western blot. The secreted form of human rpS3 rendered no endonuclease activity while the intracellular form showed UV specific endonuclease activity by the nick circle assay.
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제안 방법
In this study, the cDNA of the human rpS3 gene was subcloned in a methylotrophic yeast, Pichia pastoris, under the Pichia expression system, and the expressed protein was characterized for endonuclease activity by nick circle assay.
pastoris genome. The pPIC9 vector was constructed commercially r the secretion of the protein expressed from heterologous gene, and recombinant P. pastoris strains design nated as GS115-pPIC9-rpS3 were generated as described under Materials and Methods. The recombinant plasmid pPIC9-rpS3 was linearized by digestion with Sal I and transformed into the P.
대상 데이터
2). Nine clones (lanes 2-10) which had the integrated rpS3 gene were identified. pPIC9-rpS3 plasmid (lane 11) and chromosomal DNA from GS115-pPIC9 (lane 12) were used as positive and negative controls, respectively.
이론/모형
Control GS115-pPIC9 cells grown under the same conditions did not produce secreted rpS3 proteins in this,molecular weight Tange. The protein concentrations of GS115-pPIC9-rpS3 and GS115-pPIC9 were measured by Bradford method. Different protein quantities of GS115-pPIC9-rpS3 (12.
성능/효과
The resulting linear DNAs were used to transform GS115 according to the Lithium Chloride transformation method as previously described (3). By patching on a minimal dextrose (MD; YNB 1.34%, biotin 410-5%, dextrose 1.0%) plate and a minimal methanol (MM; YNB 1.34%, biotin 4IO-5%, methanol 0.5%) plate, Mut+ phenotype transformants were readily distinguished from Muts phenotype transformants (7). GS115/albumin (His+Mut+) on MD and MM plates were used as a control for selection of the Mut+ phenotype.
UV specific activity was calculated by subtracting the endonuclease activity of non-UV irradiated DNA from that of UV irradiated DNA. From these results, it is predicted that pPIC9 vector secretes some of heterologously expressed human rpS3 proteins into the medium (Fig. 3, lanes 2-6; Fig. 4, lanes 4 and 8), and these secreted proteins lose the endonuclease activity in the medium as murine rpS3 loses specific activity upon purification and storage (Tables 1 and 2).
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